Protein production systems, in which polypeptides or proteins of interest are produced in recombinant organisms or cells, are the backbone of commercial biotechnology.
The earliest systems, based on bacterial expression in hosts such as E. coli, have been joined by systems based on eukaryotic hosts, in particular mammalian cells in culture, insect cells both in culture and in the form of whole insects, and transgenic mammals such as sheep and goats.
Prokaryotic cell culture systems are easy to maintain and cheap to operate. However, prokaryotic cells are not capable of post-translational modification of eukaryotic proteins. Moreover, many proteins are incorrectly folded, requiring specific procedures to refold them, which adds to the cost of production.
Eukaryotic cell culture systems have been described for a number of applications. For example, mammalian cells are capable of post-translational modification, and generally produce proteins which are correctly folded and soluble. The chief disadvantages of mammalian cell systems include the requirement for specialised and expensive culture facilities, the risk of infection, which can lead to loss of the whole culture, and the risk of contaminating the end product with potentially hazardous mammalian proteins. Insect cells are alternatively used for polypeptide expression. The most widespread expression system used in insect cells is based on baculovirus vectors. A baculovirus expression vector is constructed by replacing the polyhedrin gene of baculovirus, which encodes a major structural protein of the baculovirus, with a heterologous gene, under the control of the strong native polyhedrin promoter. Cultured insect host cells are infected with the recombinant virus, and the protein produced thereby can be recovered from the cells themselves or from the culture medium if suitable secretion signals are employed.
Both systems, however, suffer from problems associated with reproducibility of recombinant protein expression level and quality, infection of the culture, and may require specialised culture facilities. Furthermore, baculovirus stocks, which for the production of certain proteins may have to be made under GMP conditions, are not always stable over time.
Drosophila cells, in particular Drosophila melanogaster S2 cells, for protein expression have been disclosed in U.S. Pat. No. 5,550,043, U.S. Pat. No. 5,681,713 and U.S. Pat. No. 5,705,359. In contrast to the Baculovirus system of the prior art, in which the protein of interest is provided only upon lysis of the infected insect cells, the method based on S2 cells provides a continuous cell expression system for heterologous proteins and therefore leads to higher expression levels.
Several other means have been suggested for enhancing the expression of heterologous protein in host cells: for example, U.S. Pat. No. 5,919,682 describes a method of overproducing functional nitric acid synthase in a prokaryote using a pCW vector under the control of tac promoter and co-expressing the protein with chaperons. Also, U.S. Pat. No. 4,758,512 relates to the production of host cells having specific mutations within their DNA sequences which cause the organism to exhibit a reduced capacity for degrading foreign products. These mutated host organisms can be used to increase yields of genetically engineered foreign proteins.
Vertebrate cells, in particular mammal cells, have also been widely used in the expression of recombinant proteins. The quantity of protein production over time from the cells growing in culture depends on a number of factors, such as, for example, cell density, cell cycle phase, cellular biosynthesis rates of the proteins, condition of the medium used to support cell viability and growth, and the longevity of the cells in culture (i.e., how long before they succumb to programmed cell death, or apoptosis). Various methods of improving the viability and lifespan of the cells in culture have been developed, together with methods of increasing productivity of a desired protein by, for example, controlling nutrients, cell density, oxygen and carbon dioxide content, lactate dehydrogenase, pH, osmolarity, catabolites, etc.
Other host cells can be used for producing heterologous recombinant proteins, notably plant cells and yeast cells.
Many pharmaceutical proteins of mammalian origin have been synthesized in plants. These range from blood products, such as human serum albumin for which there is an annual demand of more than 500 tonnes, to cytokines and other signalling molecules that are required in much smaller amounts. Most plant-derived proteins have been produced in transgenic tobacco and extracted directly from leaves. Generally, these proteins are produced at low levels, typically less than 0.1% of the total soluble protein. This low level of production probably reflects a combination of factors, with poor protein folding and stability among the most important. More recently, the tobacco chloroplast system has been used to express human proteins at much higher levels (MA JKC et al, 2004).
Yeast systems have been a staple for producing large amounts of proteins for industrial and biopharmaceutical use for many years. Yeast can be grown to very high cell mass densities in well-defined medium. Recombinant proteins in yeast can be over-expressed so the product is secreted from the cell and available for recovery in the fermentation solution. Proteins secreted by yeasts are heavily glycosylated at consensus glycosylation sites. Thus, expression of recombinant proteins in yeast systems historically has been confined to proteins where post-translations glycosylation patterns do not affect the function of proteins. Several yeast expression systems are used for recombinant protein expression, including Sacharomyces, Scizosacchromyces pombe, Pichia pastoris and Hansanuela polymorpha. Recently, a novel system with the capability of producing recombinant glycoproteins in yeast has emerged with glycosylation sequences similar to secreted human glycoproteins produced in mammalian cells. The glycosylation pathway of Pichia pastoris was modified by eliminating endogenous enzymes, which add high mannose chains to N-glycosylation intermediates. In addition, at least five active enzymes, involved in synthesizing humanized oligosaccharide chains, were specifically transferred into P. pastoris. The ability to produce large quantities of humanized glycoproteins in yeast offer advantages in that glycosylated structures could be highly uniform and easily purified. In addition, cross-contamination with mammalian viruses and other mammalian host glycoproteins may be eliminated by using fed-batch production in yeast with much shorter fermentation times than mammalian cells.
However, by using these systems, heterologous proteins are produced at approximately 1-2 mg/L in the supernatant of the cultured cells, what is quite low as compared to the goals of industrial production.
There is thus an urgent need of providing a system enabling to reach significantly higher level of heterologous protein expression.
The present invention answers this need and provides protein expression methods reaching a production level until 100 times higher than the existing means of protein production (that is, until 200 mg/L of proteins in the supernatant).
The present inventors have indeed demonstrated that the use of a nucleotide vector encoding a protein derived from the human 6-methylguanine-DNA-methyltransferase (hMGMT) protein, said hMGMT derived protein being linked, directly or not, with a protein of interest enhances the production of said protein of interest to a yield of 40 mg/L to 200 mg/L in average.